View our top tips for finding the optimal combination of Western blotting products to detect and analyze low-abundance proteins.
Detecting very low quantities of protein using Western blotting is certainly within the capabilities of the method. But in sensitive analyses, researchers can face challenges relating to the amount of sample available, primary antibody, and protein expression.
There are products that can help overcome these challenges, but it isn’t always clear which ones to use.
In this blog, we look at the properties to look for in products that support detection of low-abundance proteins. How can they be used in combination to improve the reliability of sensitive analyses?
Achieving excellent Western blotting resultsThere’s no question that researchers refine the quality and consistency of their Western blotting results as they gain experience in preparing, handling, and analyzing protein samples.
The selection of products for each step of Western blotting can also contribute to achieving high-quality results. For example, some product combinations might be well-suited to low-cost routine analyses while others might better support quantitative analyses.
So although using whatever happens to be on the shelf or in the fridge might be convenient, these products might not get you the quality or consistency you need. Figure 1 shows a summary of different product combinations you might want to use in three types of analysis.
Fig 1. A comprehensive range of options is available for each step of the Western blotting process. Choosing the optimum combination for your specific application and challenges can help to ensure you achieve your analysis goals.
For sensitive analysis, an optimal product combination maximizes the ability to detect low-abundance proteins while keeping background as low as possible. So let’s take a look at what makes these products well-suited to each stage of Western blotting.
It’s useful to make sure your protein of interest doesn’t run off the gel, or there won’t be anything to detect! This is especially so when resolving small proteins, where there is a small margin of error for run time.
Molecular weight markers containing both colored and tagged recombinant proteins enable you to monitor migration in electrophoresis and later check that your proteins have transferred successfully from gel to membrane.
Using these types of versatile markers, low-abundant proteins of interest can be observed in the electrophoresis gel, and are detectable alongside the markers on X-ray film or CCD digital images.
2. Protein transfer
Transfer is a critical step with low-abundance proteins. Any loss in transfer efficiency reduces the likelihood that you’ll be able to detect your target protein.
Using a polyvinylidene difluoride (PVDF) membrane, provides high protein binding capacity compared to nitrocellulose (NC) membranes . A small pore size also helps maximize surface area for binding, and reduces the risk of blow-through for small proteins.
The high sensitivity of PVDF membranes makes nonspecific background a potential challenge. In another post, we provide several tips on optimizing transfer that should help with sensitive analysis, and provide a comparison of the most common transfer methods.
3. Probing and detection
Maximizing the chances of target protein detection while minimizing background depends on several components: blocking agent, antibodies, and detection reagent.
Many labs use BSA or milk powder as blocking agents. When you need high sensitivity in either chemiluminescence or fluorescence detection, we recommend trying a dedicated blocking agent that reduces nonspecific binding and minimizes background noise.
For labeling, highly species-specific secondary antibodies, such as ECL HRP-conjugated antibodies, minimize nonspecific signal in chemiluminescence detection. In this case, the secondary antibody is also optimized for use with the ECL Western blotting detection reagents.
The most sensitive detection reagent in the ECL range is well-suited to sensitive analyses of low-abundance proteins. This reagent can detect the smallest of changes in protein expression, also allowing you to use highly diluted antibodies.
Choosing probing and detection reagents from the same family—for example, ECL—makes sure each component is compatible with the others and contributes to the end goal. In our 5 pain points in detection post, we also provide some troubleshooting tips to help with several potential challenges at this step.
When following recommendations for chemiluminescent detection of low-abundance proteins, there are two options for imaging:
For sensitive analysis, using X-ray film is a straightforward method for taking long exposures for low-abundance proteins. Digital CCD camera-based imaging, however, provides more control over imaging sensitivity and background correction than film.
Each imaging option has its own benefits and drawbacks, which you can discover in our in-depth piece on X-ray films and digital imaging.
Download the Western blotting principles and methods handbook how-to hints and tips, protocols, and much more.